Metastasis models using green fluorescent protein (GFP) as a marker

ABSTRACT

A method to follow the progression of metastasis of a primary tumor, which method comprises removing fresh organ tissues from a vertebrate subject which has been modified to contain tumor cells that express GFP and observing the excised tissues for the presence of fluorescence is disclosed. The fluorescence can also be monitored by observing the tissues in situ. Vertebrate subjects which contain GFP producing tumors are useful models to study the mechanism of metastasis, as well as to evaluate candidate protocols and drugs. In addition, subjects already harboring tumors can be treated so as to modify the endogenous tumors to contain GFP. This permits clinical applications. Finally, by injecting a contrast dye into a subject harboring a GFP-labeled tumor, angiogenesis in the tumor can be observed directly.

This application is a Continuation of U.S. Ser. No. 09/226,856, filedJan. 7, 1999, now U.S. Pat. No. 6,251,384, which is aContinuation-in-part of U.S. Ser. No. 09/067,734, filed Apr. 28, 1998,now U.S. Pat. No. 6,235,968, which is a Continuation-in-part of U.S.Ser. No. 09/049,544, filed March 27, 1998, now U.S. Pat. No. 5,235,967,which is a Continuation-in-part of U.S. Ser. No. 08/848,539, filed April28, 1997, now U.S. Pat. No. 6,232,523, the contents of which areincorporated by reference.

TECHNICAL FIELD

The invention relates to the study of tumor progression. Specifically,it concerns model systems for studying the metastasis of tumors invertebrate systems and to models and methods for evaluating candidatedrugs.

BACKGROUND ART

It has long been recognized that the ability of tumor tissues tometastasize constitutes a major portion of the life-threatening aspectsof malignancy. Metastasis is the growth of secondary tumors at sitesdifferent from the primary tumor. Thus, despite surgical removal of theprimary tumor, it may not be possible to arrest the progress of thiscondition. An understanding of the mechanism whereby metastasis occurswill be crucial to the development of protocols whereby the growth ofsecondary tumors can be controlled. In order to understand the mechanismof metastasis, it will be necessary to provide a model which permitsidentification of small numbers of tumor cells against a background ofmany host cells so that secondary tumor emboli and micrometastases canbe observed over the course of real time.

Others have demonstrated extravasation and initial seeding steps intumor metastasis in vitro using externally fluorescently labeled tumorcells. Khokha, R. et al., Cancer Metastasis Rev (1995) 14:279-301; Koop,S. et al., Cancer Res (1995) 55:2520-2523. Further, Margolis, L. B. etal., In Vitro Cell Dev Biol (1995) 31:221-226 was able to visualize themigration of externally fluorescently labeled lung tumor cells in hostmouse lung in histoculture. In all cases, however, long-term observationwas not possible due to the limitation of exogenous fluorescent labels.Retroviral transfer of a green fluorescent protein (GFP) gene has beenshown to result in stable transfectants of human cancer cells in vitro(Levy, J. P. et al., Nature Biotechnol (1996) 14:610-614), as well as ofhematopoietic cells (Grignani, F. et al. Cancer Res (1998) 58:14-19 andby Cheng, L. et al. Gene Therapy (1997) 4:1013-1022).

Attempts have been made to provide such a model using theβ-galactosidase gene as a marker (Lin, W. C. et al., Cancer Res (1990)50:2808-2817; Lin, W. C. et al., Invasion and Metastasis (1992)12:197-209). However, this marker has not proved satisfactory, as freshor processed tissue cannot be used. The present invention provides amarker which permits visualization of tumor invasion and micrometastasisformation in viable fresh tissue. In addition, by providing suitablecontrast media, the method of the invention can be adapted to visualizeangiogenesis in established and growing tumors. The methods of theinvention can be applied not only to models of tumor growth andmetastasis, but, through the use of retroviral vectors, can be employedto obtain clinical data in human subjects bearing tumors.

The present invention utilizes green fluorescent protein (GFP) as amarker. Heterologous expression of this protein, principally to monitorexpression of fused DNA, was disclosed in U.S. Pat. No. 5,491,084. Thisdocument describes the expression of GFP in E. coli and C. elegans andpostulates that cells in general can be modified to express GFP. Suchexpression, according to this document, permits not only a method tomonitor expression of fused DNA, but also a means of monitoring proteinlocalization within the cell.

The aspect of the invention which provides a metastatic model has beenreported and described in a series of publications. Chishima, T. et al.Cancer Research (1997) 57:2042-2047 describe the construction of adicistronic vector containing the gene for humanized green fluorescentprotein (GFP) and dihydrofolate reductase (DHFR). This vector wastransfected into CHO-K1 cells to obtain clone-38. Clone-38 showed stableGFP expression which was maintained in the presence of methotrexate(MTX). Clone-38 cells were injected into mice to obtain tumor fragmentswhich were then implanted by surgical orthotopic implantation (SOI) onthe ovarian serosa in nude mice. Metastasis could be followed in thismodel.

Chishima, T. et al. Proc Natl Acad Sci USA (1997) 94:11573-11576describe the preparation of clone-26 by transfection of Anip 973 humanlung adenocarcinoma cells with the codon optimized hGFP-S65T cloneobtained from Clontech. Clone-26 was injected intravenously into nudemice and the resulting tumors were followed in histoculture.

Chishima, T. et al. Clin Exp Metastasis (1997) 15:547-552 and Chishima,T. et al. Anticancer Res (1997) 17:2377-2384 describe similar work withclone-26 wherein the cells were inoculated subcutaneously into nude miceresulting in a visualizable tumor which was then implanted into thevisceral pleura of nude mice by SOI. Metastases were observed in thismodel as well.

Chishima, T. et al. In Vitro Cell Dev Biol (1997) 33:745-747 describehistoculture of clone-26 and visualization of growth using thefluorescence emitted by GFP.

Yang, M., et al., Cancer Res (1998) 58:4217-4221 describe transductionof the human lung cancer cell line H460 with a retroviral expressionvector containing enhanced GFP to obtain a stable high-GFP-expressingclone. Cells from this cell line were injected into nude mice and theresulting subcutaneously growing labeled tumors were transplanted by SOIinto the left lung of nude mice. Fluorescence could then be observedfrom the metastases in the collateral lung, pleural membrane andthroughout the skeletal system.

Yang, M., et al., Cancer Res (In Press) report similar studies using amodel for prostate tumor and showing fluorescence throughout theskeletal system in nude mice.

The contents of the foregoing publications are incorporated herein byreference.

In addition to the foregoing, Cheng, L., et al., Gene Therapy (1997)4:1013-1022, describe the modification of hematopoietic stem cells usingthe GFP gene under control of a retroviral promoter. Although theauthors state that human stem cells are transfected with this systemonly with difficulty, by using an enhanced form of the GFP, satisfactorybrightness could be achieved.

In addition, Grignani, F., et al., Cancer Res (1998) 58:14-19, reportthe use of a hybrid EBV/retroviral vector expressing GFP to effecthigh-efficiency gene transfer into human hematopoietic progenitor cells.

Vectors containing various modified forms of GFP to provide variouscolors are marketed by Clontech. The Clontech vectors intended formammalian cell expression place the GFP under control of thecytomegalovirus (CMV) promoter.

DISCLOSURE OF THE INVENTION

The invention provides models which permit the intimate study offormation of metastases from primary tumors in a realistic and real-timesetting. By using green fluorescent protein (GFP) as a stable andreadily visualized marker, the progression of such metastasis can bemodeled and the mechanism elucidated.

Thus, in one aspect, the invention is directed to a method to follow theprogression of metastasis of a primary tumor, which method comprisesremoving fresh organ tissues from a vertebrate subject which has beenmodified to contain tumor cells that express GFP and observing theexcised tissues for the presence of fluorescence.

In one embodiment, however, it is unnecessary to remove organ tissues;rather, the fluorescence can be visualized in the whole animal byreal-time fluorescence optical tumor imaging (FOTI).

In another aspect, the invention is directed to a vertebrate subjectwhich has been modified to contain tumor cells expressing GFP.

In these aspects, the vertebrate subject may constitute a model system,such as an immunocompromised mouse wherein tumor cells or a tumor,modified to express green fluorescent protein has been introduced intothe subject. The model system may be used to evaluate candidate drugsfor their capacity to inhibit metastasis. Alternatively, the subject maybe a human or other vertebrate which natively contains the tumor, butwherein the tumor has been subjected to viral infection or totransfection with a retroviral vector so as to produce said GFP. Theefficacy of drugs administered to such patients can be evaluated byfollowing the course of metastasis in the subject.

In still other aspects, the invention is directed to tumor cellsmodified to produce GFP under control of heterologous control elements,to cells that are immortalized to provide stable cell lines as well ascomprising visible amounts of GFP, to tissues containing metastatictumors that produce GFP, and to histocultures of tissues which containsuch metastasized tumors.

The invention also includes a method to observe and follow angiogenesisin solid tumors which method comprises (usually) exposing and observingsaid tumors. The tumors will have been modified to express GFP, and thesubject will have been administered a contrast dye to permit thisobservation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1 b show the construction of expression vectors useful inthe invention.

MODES OF CARRYING OUT THE INVENTION

The invention provides model systems for the study of the mechanism ofmetastasis of tumors generally, as well as to study angiogenesis insolid tumors. Advantage is taken of the visible marker greenfluorescence protein (GFP) to label the tumor cells so that theirmigration and colonization in tissues distal to the tumor can befollowed as the migration and colonization progresses. Further, byadministering to the subject a contrast dye, such as rhodamine, thegrowth of blood vessels in solid tumors which have been labeled with GFPcan also be observed.

Since sufficient intensity can be achieved to observe the migration offluorescent cells in the intact animal, in addition to determining themigration of the cells by excising organs, the progression of metastasiscan be observed in the intact subject. Either or both methods may beemployed to observe metastasis in evaluating, in model systems, theefficacy of potential antimetastatic drugs. The success or failure oftreatments provided to patients with potentially metastatic cancers canalso be followed using the materials and methods of the invention.

The label used in the various aspects of the invention is greenfluorescent protein (GFP). The native gene encoding this protein hasbeen cloned from the bioluminescent jellyfish Aequorea victoria (Morin,J. et al., J Cell Physiol (1972) 77:313-318). The availability of thegene has made it possible to use GFP as a marker for gene expression.GFP itself is a 283 amino acid protein with a molecular weight of 27 kD.It requires no additional proteins from its native source nor does itrequire substrates or cofactors available only in its native source inorder to fluoresce. (Prasher, D.C. et al., Gene (1992) 111:229-233;Yang, F. et al., Nature Biotechnol (1996) 14:1252-1256; Cody, C. W. etal., Biochemistry (1993) 32:1212-1218.) Mutants of the GFP gene havebeen found useful to enhance expression and to modify excitation andfluorescence. GFP-S65T (wherein serine at 65 is replaced with threonine)is particularly useful in the invention method and has a singleexcitation peak at 490 nm. (Heim, R. et al., Nature (1995) 373:663-664);U.S. Pat. No. 5,625,048. Other mutants have also been disclosed byDelagrade, S. et al., Biotechnology (1995) 13:151-154; Cormack, B. etal., Gene (1996) 173:33-38 and Cramer, A. et al. Nature Biotechnol(1996) 14:315-319. Additional mutants are also disclosed in U.S. Pat.No. 5,625,048. By suitable modification, the spectrum of light emittedby the GFP can be altered. Thus, although the term “GFP” is used in thepresent application, the proteins included within this definition arenot necessarily green in appearance. Various forms of GFP exhibit colorsother than green and these, too, are included within the definition of“GFP” and are useful in the methods and materials of the invention. Inaddition, it is noted that green fluorescent proteins falling within thedefinition of “GFP” herein have been isolated from other organisms, suchas the sea pansy, Renilla reriformis. Any suitable and convenient formof the GFP gene can be used to modify the tumor cells useful in themodels of the invention, and for retroviral transformation of endogenoustumors. The particular humanized hGFP-S65T clone is used in the examplesset forth below for illustration.

Techniques for labeling cells in general using GFP are disclosed in U.S.Pat. No. 5,491,084 (supra).

In one application, the method of the invention provides a model systemfor studying the effects of various therapeutic candidate protocols andsubstances on metastatic growth of tumors.

In general, the model involves modifying a vertebrate, preferably amammal, so as to contain tumor tissue, wherein the tumor cells have,themselves, been modified to contain an expression system for GFP. Thetumor cells may arise from cell lines of the invention wherein tumorcells have been modified to contain expression systems for GFP and SV40T-antigen. Tumors can be formed in such vertebrate systems byadministering the transformed cells containing the GFP expression systemand permitting these transformed cells to form tumors. Typically suchadministration is subcutaneous and the tumors are formed as solidmasses. The tumors thus formed can be implanted in any suitable hosttissue and allowed to progress, metastasize and develop.

Suitable procedures for growing the initial tumor, thus, involvetranscutaneous injection of the tumor cells producing GFP, such as CHOcells, HeLa cells, carcinoma and sarcoma cell lines, well establishedcell lines such as the human lung adenocarcinoma line Anip 973, or lungcancer cell line H460 as well as GFP-containing human breast cancerlines MDA-MB468 and MDA-MB435; human prostate cancer lines PC3 andDU-145, human glioblastoma line 324, mouse melanoma B16 and others thatmay become available in the art, including the immortalized cells of theinvention. The administered cells will have been modified to contain anexpression system for GFP. After administration, solid tumors generallydevelop, typically at the site of subcutaneous injection. These tumors,which are themselves fluorescent, can then be removed and used forimplantation in the model vertebrate.

Techniques for implantation of the solid tumors, now labeled with GFP,into vertebrates include direct implantation by surgical orthotopicimplantation (SOI) at the desired site, typically the site from whichthe tumor cells were derived. Suitable sites include lung, liver,pancreas, stomach, breast, ovary, prostate, bone marrow, brain, andother tissues susceptible to malignancy. Once the solid tumors have beenimplanted, the vertebrate becomes a model system for studyingmetastasis. The tumor is thus allowed to progress and develop and thevertebrate is monitored for appearance of the GFP labeled cells at sitesdistal from the original implantation site. The monitoring can occureither on the whole vertebrate by opening the animal and observing theorgans directly with a fluorescent microscope, or the tissues may beexcised and examined microscopically. In some cases the tumors aresufficiently bright that opening the animal is unnecessary—they can beseen directly through the skin. In any case, as GFP is visible to thenaked eye, no development systems to stain the tissue samples arerequired. Tissue samples are simply properly processed as fresh samplesin slices of suitable size, typically 1 mm thick, and placed under amicroscope for examination. Even colonies of less than 10 cells are thusvisible. A variety of microscopic visualization techniques is known inthe art and any appropriate method can be used.

It is particularly convenient to visualize the migration of tumor cellsin the intact animal through fluorescent optical tumor imaging (FOTI).This permits real-time observation and monitoring of progression ofmetastasis on a continuous basis, in particular, in model systems, inevaluation of potential anti-metastatic drugs. Thus, the relative lackof metastasis observed directly in test animals administered a candidatedrug in comparison to controls which have not been administered thedrugs indicates the efficacy of the candidate and its potential as atreatment. In subjects being treated for cancer, the availability ofFOTI permits those devising treatment protocols to be informed on acontinuous basis of the advisability of modifying or not modifying theprotocol.

In addition, the development of the tumor can be studied in vitro inhistological culture. Suitable systems for such study include solidsupported cultures such as those maintained on collagen gels and thelike.

Suitable vertebrate subjects for use as models are preferably mammaliansubjects, most preferably convenient laboratory animals such as rabbits,rats, mice, and the like. For closer analogy to human subjects, primatescould also be used. Particularly useful are subjects that areparticularly susceptible to tumor development, such as subjects withimpaired immune systems, typically nude mice or SCID mice. Anyappropriate vertebrate subject can be used, the choice being dictatedmainly by convenience and similarity to the system of ultimate interest.

Any suitable expression system operable in the tumor cells to beimplanted may be used. A number of vectors are commercially availablethat will effect expression in tumor cells of various types. The natureof the vector may vary with the nature of the tumor and the vertebratein which it finds its origin. However, when GFP is used to visualizemetastasis in a model system, it is preferred to utilize vectors whichdo not use retroviral or other viral promoters which may complicate thenature of the model.

In order to provide cell lines that are helpful in establishing tumorsfor these model systems, it is also advantageous to employ expressionvectors which provide the cells with the SV40 T-antigen. The presence ofthis antigen ensures immortality of the culture. Thus, particularlyuseful in the invention are vectors which comprise expression systemsthat result in the production both of GFP and SV40 T-antigen.

In order to transfect and modify the transformed cells which areeffective in generating tumors, any suitable transfection method may beused, such as liposomes, calcium phosphate precipitation,electroporation and use of a gene gun. Lipofection is preferred.

In contrast, when the method of the invention is used to visualizemetastasis in tumors that natively occur in a subject such as a humancancer patient, vectors that employ retroviral or other viral promotersare preferred. The use of such vectors permits the insertion of anexpression system for GFP into the already existent tumor. In addition,the expression system may contain nucleotide sequence encoding otheruseful proteins such as therapeutic proteins which permit simultaneousdiagnosis of metastasis and treatment. Among such suitable proteins areincluded methioninase (see, for example, PCT/US93/11311 andPCT/US96/09935). Such proteins may be produced either as fusions withthe GFP, or independently either using a dicistronic expression systemor independent expression systems, one for the therapeutic protein andthe other for the GFP.

Retroviral based expression systems for GFP have already been describedby Grignani, F. et al. Cancer Res (1998) 58:14-19 and by Cheng, L. etal. Gene Therapy (1997) 4:1013-1022. In these reports, the retroviralexpression system itself was used to transfect hematopoietic progenitorcells or packaging cells were employed to provide virus-containingsupernatants which can be used directly for infection of the mammaliancells. Thus, in the method of the invention, the tumor contained in thevertebrate subject is typically infected with virus which has beenmodified and packaged to contain the expression system for GFP. In situinfection with virus results in the ability of the tumor to produce GFPand, in effect, label itself.

Various retroviral systems useful in producing proteins in mammaliancells are known in the art. Examples include commercially availablevector and packaging systems such as those sold by Clontech, San Diego,Calif., including their Retro-X vectors pLNCX and pLXSN which permitexpression of GFP under a variety of promoters by insertion into themultiple cloning site. These vectors contain ψ* (the extended viralpackaging signal) and antibiotic resistance genes for selection. Anumber of these systems have been developed for use in gene therapy,including vectors which provide a multiple cloning site sandwichedbetween 5′ and 3′ LTR derived from retroviral sources, and thus would beuseful in labeling the tumors of human patients.

Thus, retroviral based vectors such as those set forth in FIGS. 1a-1 bcan be transfected into packaging cells and transferred directly totargeted cancer cells or supernatants from the packaging cells can beused to infect tumor cells with the retrovirus. Preferred combinationsof retrovirus and packaging cells include the GFP-retrovirus vectorpLEIN in PT-67 packaging cells. Co-culture of the packaging cells withcolon cancer cells results in transfer of the GFP-retrovirus to thecancer cells.

Using histoculture techniques, and supernatants from PT-67 packagingcells generating GFP-pLEIN virus, the successful modification of a humancancer tissue to display the fluorescence associated with GFP has beendemonstrated. For use in vivo, the virus is administered, preferablylocally to the tumor, which can be observed within hours after injectioneither of packaging cells or of the viral containing supernatants. Themalignant cells can be identified by their green color, sometimessufficiently bright so that the tumors can be seen through the skin.

In addition to direct observation of tumor metastasis and growth eitherin a model system or in a vertebrate, typically mammalian and moretypically a human subject which is already afflicted by a tumor, themethods of the invention can be adapted to observe angiogenesis in solidtumors. The tumor is itself labeled with GFP as described above. Thesubject is then administered a contrast dye, typically by injection,preferably intravenous injection, which allows blood vessels in thetumor to be observed. Suitable dyes include rodamine and other contrastdyes. Any dye which forms a contrasting color with the green color ofthe GFP can be used. Preferably, the dye is coupled to an inert polymersuch as polyethylene glycol to increase the length of time the dye willremain in the blood vessel. A sufficient amount of dye is provided topermit ready visualization; the amount of dye required will depend onthe choice of dye, the location of the tumor, the nature of thebackground GFP, and the method used for observation. Within a fewminutes, vessels growing into the solid tumors in such areas as themesentery, colon wall, and omentum can be observed. Observations can becontinued over substantial periods; for example, angiogenesis afterseveral hours is still observed by using this method.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of Tumor Cells that Produce GFP

The humanized hGFP-S65T clone described by Zolotukhin, S. et al., JVirol (1996) 70:4646-4654 was used as the green fluorescent proteincoding sequence. This codon-optimized gene was purchased from ClontechLaboratories, Inc. (Palo Alto, Calif.) and ligated into the dicistronicexpression vector (pED-mtx¹) obtained from Genetics Institute,Cambridge, Mass. and described in Kaufman, R. J. et al., Nucleic AcidsRes (1991) 19:4485-4490. hGFP-S65T was digested with HindIII andblunted; the entire hGFP coding region was excised with XbaI and thenunidirectionally subcloned into pED-mtx¹ which had been digested withPstI, blunted and then further digested with XbaI.

CHO-K1 cells were cultured in DMEM containing 10% fetal calf serum, 2 mML-glutamine and 100 μM nonessential amino acids. Near confluent cellswere incubated with a precipitated mixture of LipofectAMINE™ reagent(GIBCO) and saturating amounts of plasmids for six hours and thenreplenished with fresh medium. The cells were harvested by trypsin/EDTA48 hours later and subcultured at 1:15 into selective medium containing1.5 μM methotrexate (MTX). Cells with stably integrated plasmids wereselected in MTX-containing medium and isolated with cloning cylinders(Bel-Art Products, Pequannock, N.J.) by EDTA. After amplification andtransfer, Clone-38 was selected because of its high-intensity GFPfluorescence and stability.

In a similar manner, Anip 973 cells, a human lung cancer cell lineobtained from Harbin Medical University, China, were cultured asdescribed above for CHO-K1 cells except using RPMI1640 (GIBCO) in placeof DMEM. Transfection, selection and amplification and transfer wereconducted as described above. Clone-26 was chosen because of itshigh-intensity GFP fluorescence and stability.

EXAMPLE 2 Mouse Model Using Modified CHO Cells

Clone-38, which was stable at 1.5 μM MTX and which proliferated at thesame rate as the parental CHO-K1 cells as ascertained by comparingdoubling times, was used in this model.

Three six-week old Balb/C nu/nu female mice were injected subcutaneouslywith a single dose of 10⁷ Clone-38 cells that had been harvested bytrypsinization and washed three times with cold serum-containing mediumand then kept on ice. The cells were injected in a total volume of 0.4ml within 40 minutes of harvesting and the nude mice sacrificed threeweeks after injection. All of the mice had a subcutaneous tumor rangingin diameter from 13.0 mm to 18.5 mm (mean=15.2 mm±2.9 mm). The tumortissue was strongly fluorescent. It was shown by extracting GFP fromcultured Clone-38 cells in comparison to Clone-38 cells prepared fromthe tumor that the levels of production of GFP were the same in both.

To construct the model, tumor fragments (1 mm³) derived from the nudemouse subcutaneous Clone-38 tumor grown as described above, wereimplanted by surgical or surgical orthotopic implantation (SOI) on theovarian serosa in six nude mice as described by Fu, X. et al.,Anticancer Res (1993) 13:283-286, incorporated herein by reference.Briefly, the mice were anesthetized by isofluran inhalation and anincision was made through the left lower abdominal pararectal line andperitoneum to expose the left ovary and part of the serosal membrane,which was scraped with a forceps. Four 1 mm³ tumor pieces were fixed onthe scraped site with an 8-0 nylon suture and the ovary then returned tothe peritoneal cavity. The abdominal wall and skin were closed with 6-0silk sutures.

Four weeks later, the mice were sacrificed and lung and various otherorgans were removed. The fresh samples were sliced at approximately 1 mmthickness and observed directly under fluorescent and confocalmicroscopy. Samples were also processed for histological examination forfluorescence and conventional staining. Frozen sections were preparedwherein the slides were rinsed with phosphate buffer saline and fixedfor 10 minutes at 4° C.; 10% formaldehyde plus 0.2% glutaraldehyde andPBS were added and the slides were then washed with PBS. The fixedtissue was stained with hematoxylin and eosin using standard techniques.

Light and fluorescence microscopy were carried out using a Nikonmicroscope equipped with a Xenon lamp power supply and a GFP filter set(Chromotechnology Corp., Brattleboro, Vt.). Confocal microscopy was withan MRC-600 Confocal Imaging System (Bio-Rad) mounted on a Nikonmicroscope with an argon laser.

The mice, at sacrifice, had tumors in the ovaries ranging in diameterfrom 18.7 mm-25.3 mm (mean 21.9±3.1 mm). The fresh organ tissuesexamined under fluorescence microscopy with no treatment of the tissuesshowed seeding of the tumor throughout the peritoneal cavity, includingthe colon (6/6 mice), cecum (5/6), small intestine (4/6), spleen (1/6),and peritoneal wall (6/6). Numerous micrometastases were detected in thelungs of all mice and multiple micrometastases were also detected on theliver (1/6), kidney (1/6), contralateral ovary (3/6), adrenal gland(2/6), para-aortic lymph node (5/6) and pleural membrane (5/6).Single-cell micrometastases could not be detected by the standardhistological techniques described above and even the multiple cellcolonies were difficult to detect using them. As the colonies developed,the density of tumor cells decreased markedly in the center.

In an additional experiment, 5×10⁶ Clone-38 cells were injected into anude mouse through the tail vein and the mouse sacrificed after twominutes. Fresh visceral organs were analyzed by fluorescence microscopyand showed the presence of fluorescent cells in peritoneal wall vesselswhich formed emboli in the capillaries of the lung, liver, kidney,spleen, ovary, adrenal gland, thyroid gland and brain.

Thus, using these techniques, progression of micrometastasis can beobserved as seeded cells develop into colonies within the relevanttarget organs. Further, screening for micrometastases can be done easilyand quickly in all systemic organs.

EXAMPLE 3 Murine Model Using Human Lung Cancer Cells

The procedures are generally those set forth in Example 2 except thatClone-26 cells as prepared in Example 1 were used instead of Clone-38CHO cells.

A. As in Example 2, tumors were grown in six-week-old Balb/C nu/nu malemice injected subcutaneously with a single 0.4 ml dose of 10⁷ Clone-26cells within 40 minutes of harvesting by trypsinization and washingthree times with cold serum-containing medium. The cells were kept onice prior to injection. The animals were sacrificed when the tumors hadreached approximately 1.2 cm diameters. The 1.2 cm tumors formed afterabout 5 weeks.

B. The tumor pieces, 1 mm³, were implanted by SOI into the left visceralpleura of 8 mice as described by Astoul, P. et al., Anticancer Research(1994) 14:85-92; Astoul, P. J Cell Biochem (1994) 56:9-15, bothincorporated herein by reference. Briefly, the mice were anesthetized byisofluoran inhalation an a small 1 cm transverse incision made on theleft lateral chest, via the fourth intercostal space, resulting in totallung collapse. Five tumor pieces were sewn together with a 7-0 nylonsurgical suture and fixed by making one knot. The lung was taken up byforceps and the tumor sewn into the lower part of the lung with onesuture, after which the lung was returned to the chest cavity and themuscles and skin closed with a single layer of 6-0 silk sutures. Thelung was reinflated by withdrawing air from the chest cavity with a23-gauge needle.

C. Four of the mice were sacrificed at 4 weeks and another 4 at 8 weeks.Pleural tumors for the 4-week group ranged from 244.40 mm³-522.88 mm³;those from the 8 week group from 1279.08 mm³-2714.40 mm³. Thisrepresented mean volumes of 371 mm³ and 1799 mm³. Specimens of tissuewere sliced at 1 mm thickness and observed directly under fluorescentmicroscopy using a Nikon microscope equipped with a Xenon lamp powersupply and a Leica stereo fluorescence microscope equipped with amercury lamp power supply and GFP filter sets. All of the animals showedchest wall invasion and local and regional spread of the tumor, but inthe 8-week mice, all tumors involved the mediastinum and contralateralpleural cavity as well as metastases on the visceral and parietalpleura. Pulmonary hilum lymph nodes were involved in 3 of 4 mice of the4-week group and all of the mice in the 8-week group. Cervical nodeinvolvement was detected in one of the mice of the 8-week group, but noother metastases were observed. The animals were also observed directlybefore the tissues were excised. The margin of the invading tumor innormal lung tissue could be detected by GFP fluorescence and a smallvessel could be seen developing at the margin of the tumor.

D. In an additional experiment, 8 nude mice were injected in the tailvein with a single dose of 1×10⁷ Clone-26 cells that had been harvestedby trypsinization and washed 3 times with cold serum-containing medium.The injection contained a total volume of 0.8 ml within 40 min. ofharvesting. Again, 4 mice were sacrificed at 4 weeks and another 4 at 8weeks and tissue specimens were obtained and studied by microscopy asdescribed above. Numerous micrometastatic colonies were detected inwhole lung tissue in both groups ranging from 5.2 μm to 32.5 μm in the4-week group and 5.5 μm-178.3 μm in the 8-week group. The colonies fromthe 8-week group did not appear further developed as compared with thosefrom the 4-week group. Numerous small colonies ranging in number to lessthan 10 cells were detected at the lung surface in both groups and brainmetastases were detected in 1 mouse of the 4-week group and 2 from the8-week group. One mouse in the 8-week group had systemic metastases inthe brain, the submandibular gland, the whole lung, the pancreas, thebilateral adrenal glands, the peritoneum and the pulmonary hilum lymphnodes.

E. In an additional experiment, similar to that set forth in theprevious paragraph, the mice injected in a tail vein with 10⁷ Clone-26cells were sacrificed at 4, 8 and 12 weeks and the tissues examined asdescribed. Most of the colonies and mice sacrificed at 8 weeks were notobviously further developed compared with those sacrificed at 4 weeks,but numerous small quantities ranging in number down to less than 10cells and ranging in size from 5.5 μm-110 μm were detected at the lungsurface. At 12 weeks, there were many small metastatic colonies whichappeared dormant, although other colonies grew extensively by this time,reaching a size up to 1100 μM, suggesting a heterogeneity of dormant andactive tumor colonies in the lung.

EXAMPLE 4 Growth of Clone-26 Tumor Cells in Histoculture

Six-week old SCID/SCID mice were injected intravenously with a singledose of 7.5×10⁷ Clone-26 cells which had been harvested bytrypsinization and washed 3 times with cold serum-containing medium andkept on ice as described above. The cells were injected in a totalvolume of 0.5 ml within 40 minutes of harvesting. After 3 weeks,numerous micrometastatic colonies were detected in whole lung tissue upto approximately 550 μm. After 5 weeks, the mice were sacrificed and theClone-26 seeded mouse lungs were removed and histocultured on spun gelsusing the histoculture methods developed by Leighton, J. Cancer Res(1957) 17:929-941; Leighton, J. et al., Cancer Res (1960) 20:575-597;Hoffman, R. M. Cancer Cells (1991) 3:86-92. Tumor colonies spreadrapidly in the lung tissue over time and after 1 week the tumor cellsstarted to invade and colonize supporting collagen sponge-gel. After 2weeks, tumor cells formed satellite colonies in the sponge-gel distantfrom the primary colonies in the lung tissue, thus growing faster inhistoculture than in SCID mice. Tumor colonies could grow inhistoculture for more than 1 month.

EXAMPLE 5 Construction of a Retroviral Expression Vector for GFP andPreparation of Labeled Tumor Cell Lines

FIGS. 1a and 1 b show the construction of expression vectors for GFPunder control of the SV40 promoter. The constructs employ commerciallyavailable pEGFP series vectors available from Clontech. Both bacterialand mammalian expression vectors are available which permit productionof additional proteins, as well as GFP, either as fusions or indicistronic systems. FIG. 1a shows the construction of an expressionvector, pGFP/Met, for a fusion of GFP with methioninase; FIG. 1b showsthe construction of a vector pGFP/SV40 for production of a fusionprotein of GFP with the SV40 T-antigen.

Commercial vectors containing the GFP coding sequence of the desiredspectral characteristics using the pLEIN system described in Example 6were transfected into cell lines originating from tumors, such as humanbreast cancer, human prostate cancer, human glioblastoma and mousemelanoma. In this manner, human breast cancer cell lines MF-7, MDA-MB468and MDA-MB435, human prostate cancer cell lines PC3 and DU145, humanglioblastoma cell line 324, human lung cancer cells Anip-73 and H460,human colon cancer cells lines Colo-205, HCT-15 and WiDr, human gastriccancer cell line NVGC-4, human kidney cancer cell line SN12C, humantongue cancer cell line SCC-25, human melanomas LOX and SK-mel-5,labeled Chinese hamster ovary cells from cell line CHO-K1 and mousemelanoma cell line B16 labeled with green fluorescent protein wereestablished.

The SV40 T-antigen protein is useful to immortalize cultured cells so asto establish permanent cell lines. Accordingly, the vector pGFP/SV40 istransfected into a series of tumor cell cultures to provide fluorescentimmortalized cell lines.

EXAMPLE 6 In Vivo Labeling of Established Tumors

Unlabeled tumors derived from the human lung cancer cell line Anip973were established in mice using the procedure set forth in Example 3,paragraphs A and B, but substituting unlabeled Anip973 cells for clone26. The mice were then injected with 1×10⁷ packaging cells containingthe retroviral vector GFP-retrovirus pLEIN contained in PT67 cells. Thisvirus packaging system is available from Clontech, San Diego, Calif.pLEIN contains an insert of the coding sequence for EGFP, a red-shiftedvariant of wild-type GFP that has been optimized for brighterfluorescence and higher expression in mammalian cells. It has anexcitation maximum of 488 nm and an emission maximum at 507 nm. Thismutant contains a double amino acid substitution at position 64 from Pheto Leu and at position 65 from Ser to Thr. It is described by Comack, B.et al. Gene (1996) 173:31-38. There are more than 190 silent basechanges to maximize human codon usage preferences as described by Haas,J. et al. Curr Biol (1996) 6:315-324. Thus, pLEIN contains theabove-described GFP coding sequence inserted into the multiple cloningsite of pLXIN to obtain a dicistronic expression system which permitscoordinated translation of the GFP and neomycin resistance. Three daysafter injection of the cells into the peritoneal cavity of the mice, thetumor cells could be seen in the seminal vesicles under bright-fieldmicroscopy and under fluorescent microscopy.

EXAMPLE 7 Observation of Angiogenesis

A suspension containing 1×10⁷ clone-38 cells, described in Example 1,were injected into the peritoneal cavity of a mouse. Five days later,the mouse was injected in the tail with rhodamine and the mouse was thenput under anesthesia and the abdominal cavity opened sufficiently tovisualize the tumor. Recovery from this surgery is straightforward. Insome cases, abdominal opening is unnecessary as the intraperitonealtumors can be visualized through intact skin. Tumors were visible in theabdominal cavity and angiogenesis was apparent as identified by therhodamine fluorescence. Similar results were found in tumors growing inthe omentum in the wall of the small intestine, and in the mesentery.

In an analogous experiment, a suspension containing 1×10⁷ cells ofclone-26, described in Example 1, were injected into the peritonealcavity of a mouse. After one day, tumors appeared in the mesentery andin the colon wall. These were observed by anesthetizing the mouse and aminimal opening of the abdomen. Observations on day 3 of a similarlytreated mouse showed tumors in the wall of the small intestine and inthe omentum as well as in the colon wall and mesentery. On day 5, asimilarly treated mouse was injected in the tail with 100 μl of 2×10³ Mrhodamine and a few vessels could be seen in the tumor growing in themesentery. After day 60, numerous vessels were seen in the tumor growingin the colon wall.

EXAMPLE 8 Construction of Metastatic Models

Using the labeled human cancer cell lines described in Example 5, murinemodels are established for various types of cancer. The cell lines areimplanted into 6-week-old nu/nu female mice with a single dose of 107GFP expressing human tumor cells which had been harvested bytrypsinization and washed three time with cold, serum-containing mediumand then kept on ice. The cells are injected in subcutaneous space inthe flank of the animal at a total volume of 0.4 ml within 40 min ofharvesting. The nude mice are sacrificed to harvest the tumor fragments3 weeks after tumor cell injection. These tumor fragments are then usedfor surgical implantation into the corresponding tissue (surgicalorthotopic implantation (SOI)) in nude mice as recipients.

The recipient mice are first anesthetized and then implanted usingestablished SOI techniques with fragments of the subcutaneously growncolon cancer, lung cancer, breast cancer, prostate cancer or melanoma.In all cases, except for melanoma, the size of the fragment is 1 mm³;for melanoma, 0.025 mm³ fragments are prepared from the human melanomaLOX-GFP subcutaneous tumor and 5-6 fragments are implanted. The progressof metastasis is then observed using FOTI with a Leica StereomicroscopeMZ12 with a mercury lamp source. GFP is excited with a D425/60 bandpassfilter and a 470DCXR dichroic mirror; fluorescence is emitted through aGG475 longpass filter (Chroma Technology, Brattle-boro, Vt.) andcollected by a thermoelectrically cooled ST-133 Micromass High-SpeedControlled Camera—TEA/CCD-1317K1 (Princeton Instruments, Trenton, N.J.)with a 17×1035 pixels chip. The images are processed and analyzed withImagePro+3.1 Software (Media Cybernetics, Silver Spring, Md.). Highresolution images are captured by computer, or continuously throughvideo output onto video tape.

In the colon cancer model, a small midline incision is made in theabdomen and the colorectal part of the intestine is exteriorized. Theserosa is removed and 8-15 pieces of tumor fragments are implanted. An8-0 surgical suture is used to penetrate the small tumor pieces andsuture them to the wall of the intestine. The intestine is returned tothe abdominal cavity and abdominal wall is closed. The animals are thenobserved for metastases.

For lung cancer models, a small 1 cm transverse incision is made on theleft lateral chest via the fourth intercostal space; total lung collapseresults. Five tumor pieces sewn together with 8-0 nylon surgical sutureare fixed by making one knot; the lung is taken out by forceps and thetumor sewn into the lower part of the lung with one suture. Afterreturning the lung to the chest cavity, the chest muscles and skin areclosed. The lung is reinflated by withdrawing air from the chest cavitywith a 23-gauge needle. The animals can then be observed for metastasiseither by FOTI or by excising various tissues.

For breast cancer, an incision of 1.5 cm is made along the medial sideof the nipple and after blunt dissection, the fat pad is exposed. Asmall incision is made and a small pocket formed to accommodate 2fragments of the tumor tissue; an 8-0 suture is made to close thepocket. The skin layer is then closed. The animals are then observed byFOTI or by tissue excision.

For prostate cancer, an opening is made above the pubic symphysis toexpose the prostate gland. The fascia surrounding the dorsal portion ofthe prostate and the dorsal lateral lobes of the gland are separated bya small incision. Five randomized fragments are sutured into theincision using a 8-0 nylon suture. The two parts of the separated lobesare sutured together and the surrounding fascia used to wrap thisportion of the gland to consolidate the incision. The abdomen is thenclosed and the animals maintained for observation.

For melanoma, 5-6 fragments are transplanted subdermally into the flankwith a 13×¼ cancer implant needle (Popper & Sons, New Hyde Park, N.Y.).

Images can be obtained as described above showing metastases to variouslocations in the animal.

The animals treated as described above, can then be used to evaluatepotential protocols for treatment of cancer and metastasis inhibition.The metastatic progress of the fluorescent tumors in animalsadministered the protocols is compared to similar animals lackingtreatment. The efficacy of the protocols can then be directly observed.

What is claimed is:
 1. A method to evaluate a candidate protocol or drug for the inhibition of metastasis of a primary tumor which method comprises: administering said protocol or drug to a subject which is a mouse, rat or rabbit which contains a primary tumor that stably expresses green fluorescent protein (GFP) in cells of said tumor when said tumor metastasizes and monitoring the progression of metastasis by observing the presence, absence or intensity of the fluorescence at various locations in the treated subject; wherein said subject contains said tumor that expresses GFP and wherein said subject is a genetically immunocompromised mouse, rat or rabbit, or a mouse, rat or rabbit which is syngeneic to said tumor; monitoring the progression of metastasis in a control, which contains a similar tumor that expresses green fluorescent protein; wherein said control subject contains said tumor that expresses GFP wherein said control subject is an immunocompromised mouse, rat or rabbit, or a mouse, rat or rabbit which is syngeneic to said tumor; and comparing the progression of metastasis in said treated subject with the progression of metastasis in said control subject wherein the control subject and treated subject are intact; whereby a diminution of the progression of metastasis in said treated subject as compared to said control subject identifies the protocol or drug as effective in inhibiting metastasis.
 2. The method of claim 1 wherein the progression of metastasis is monitored by fluorescent optical tumor imaging in the intact subject.
 3. The method of claim 1 wherein said subject contains said tumor by virtue of surgical orthotopic implantation of said tumor.
 4. The method of claim 1 wherein said subject contains said tumor by virtue of injecting cells of a stably transformed tumor cell line which has been transfected with an expression vector containing a first nucleotide sequence encoding green fluorescent protein (GFP) and a second nucleotide sequence encoding a selection marker, both said first and second nucleotide sequences being under control of a viral promoter and wherein said cell line stably effects high level expression of said GFP in the absence of selection agent and maintains a high level expression of GFP when said cell line proliferates through multiple passages of said cell line.
 5. A method to monitor metastasis of a primary tumor in a subject which is a mouse, rat or rabbit which contains said primary tumor, and wherein said tumor stably expresses green fluorescent protein (GFP) in cells of said tumor when said tumor metastasizes, wherein said subject contains said tumor that expresses GFP and wherein said subject is a genetically immunocompromised mouse, rat or rabbit, or a mouse, rat or rabbit which is syngeneic to said tumor; which method comprises monitoring the progression of metastasis by observing the presence, absence or intensity of the fluorescence as a function of time at various locations in said subject wherein the subject is intact.
 6. The method of claim 5 wherein the progression of metastasis is monitored by fluorescent optical tumor imaging in the intact subject.
 7. The method of claim 5 wherein said subject contains said tumor by virtue of surgical orthotopic implantation of said tumor.
 8. The method of claim 5 wherein said subject contains said tumor by virtue of injecting cells of a stably transformed tumor cell line which has been transfected with an expression vector in containing a first nucleotide sequence encoding green fluorescent protein (GFP) and a second nucleotide sequence encoding a selection marker, both said first and second nucleotide sequences being under control of a viral promoter and wherein said cell line stablely effects high level expression of said GFP in the absence of selection agent and maintains a high level expression of GFP when said cell line proliferates through multiple passages of said cell line.
 9. A method to monitor metastasis of a primary tumor in a mammalian subject which contains said primary tumor, and wherein said tumor stably expresses green fluorescent protein (GFP) in cells of said tumor when said tumor metastasizes, wherein said primary tumor is endogenous to said mammalian subject and expresses said GFP as a result of locally administering a retroviral vector to said subject in the vicinity of said tumor, said retroviral vector containing an expression system for said GFP; which method comprises monitoring the progression of metastasis by observing the presence, absence or intensity of the fluorescence as a function of time at various locations in said subject.
 10. The method of claim 9 wherein the subject is human.
 11. The method of claim 9 wherein the progression of metastasis is monitored by excising fresh organ tissues from various locations in said subject.
 12. The method of claim 11 wherein said excised tissues are observed by microscopic examination of fresh tissue slices. 